(1) Filed of the Invention
The invention concerns peptide sequences, compositions comprising the peptide sequences, and in particular influenza vaccines comprising the sequences and the compositions, and uses of the sequences. The present invention is especially concerned with vaccines that are protective against a plurality of influenza virus strains, including existing viruses across different species (e.g. protective against both human and avian influenza) as well as future viruses that have mutated from existing viruses (such as a future mutated form of avian flu that is readily transmissible from human to human, which could potentially give rise to an influenza pandemic).
(2) Description of Related Art
The defense against disease is critical for the survival of all animals, and the defense mechanism employed for this purpose is the animal immune system. Understanding the immune system is therefore a key to understanding the development of new and more sophisticated treatments for humans and animals alike.
The mechanism of operation of the immune system has been under investigation for many years. The system is composed of a number of cell types and a variety of molecules, making it extremely complex. Even after many years of study, the full extent of the immune system components, and their interaction with each other, is imperfectly understood.
Many years ago it was recognised that a person who recovers from a particular disease may acquire some protection in future against that disease, but not against a disease which that person has not yet contracted. This fundamental aspect of the immune system was interpreted at that time by considering that the immune system acquired a kind of ‘memory’ against certain pathogens once exposure to such pathogens had taken place, that memory being specific to a certain disease.
Gradually, it became known that exposure to less harmful variants of a pathogen could induce protection against more harmful variants (e.g. exposure to cowpox to protect against smallpox, or exposure to an inactivated anthrax to protect against live anthrax). Thus, the idea of vaccination against a disease arose.
It is now known that the immune system has at least two divisions: innate immunity and adaptive immunity. The innate system is fully functional before a pathogen enters the system, whilst the adaptive system is switched on after the pathogen enters the system. It then develops an attack specific to the pathogen. The innate system comprises a number of components, including phagocytes such as macrophages, which (as the name suggests) ‘eat’ or engulf foreign bodies such as pathogens.
Typically, but not exclusively, the present invention is concerned with the adaptive immune system, and unless specifically indicated otherwise, ‘immune system’ in the present context refers to the adaptive immune system.
In order to understand more fully how the immune system functions, the role of its individual components must be carefully considered. In respect of the adaptive immune system, it is well known that immunity against pathogens is provided by the action of lymphocytes, which constitute the most common cell type in the immune system. There are two types of lymphocyte: the B lymphocyte and the T lymphocyte. These are generally termed B cells and T cells respectively.
B cells have the ability to develop into plasma cells, which manufacture antibodies. Antibodies are very important components of the animal immune system. They are produced in response to some signature portion of the invading pathogen (an antigen of the pathogen—antigens here being defined as any foreign substance recognised by the immune system) and are usually specific to that pathogen. However, if two pathogens are very similar, or at least contain the same antigen, then antibodies produced against one can nevertheless be effective against the other (they may ‘cross-react’). This explains why inoculation with cowpox may protect against smallpox. It is important to realise that the antibodies ‘recognise’ only a small portion of the antigenic molecule of the pathogen rather than the pathogen as a whole. These portions are termed epitopes.
T cells do not possess or produce antibodies. Instead, they recognise fragments (i.e. epitopes) of the foreign antigen complexed with major histocompatibility complex (MHC) (or in the case of humans, human leucocyte antigen (HLA)) via a specialised receptor known as TCR (T cell receptor). T cells are themselves divisible into subsets which can have either a regulatory function or an effector function. The effector cells are involved with ‘effecting’ the removal of foreign substances. For example, cytotoxic T cells (CTL) are effector cells that are able to kill infected cells, as well as other unwanted species such as tumour cells. Regulatory T cells, on the other hand, play a role in helping effector T and B cells to become more effective. Due to this function, these regulatory T cells are often termed ‘helper’ T cells. Other regulatory T cells, termed ‘suppressor’ T cells, are thought to inhibit immune responses, but these are less well understood. Regulatory T cells may also interact with components of the innate immune system to boost their activity.
In a normal healthy individual, the lymphocytes in the immune system remain in an inactive ‘resting’ state until an immune response is triggered. When an immune response is required, the lymphocytes become activated, proliferate and begin to carry out their designated functions. For example, any resting T cell displaying on its surface a TCR that recognises an epitope of the invading pathogen complexed with a MHC molecule is activated, proliferates (this being termed clonal expansion) and the resulting offspring start to actively carry out their predetermined effector functions required to combat the invading organisms.
When the immune response is completed, (i.e. the pathogens and/or infected cells have been eliminated) the lymphocytes revert to a resting state once again. This resting state is not, however, equivalent to the initial inactive resting state. Activated, but resting lymphocytes, can be rapidly recruited and induced to proliferate in response to an infection by the same, or closely related, pathogen at a later time.
This ability of activated resting lymphocytes, to deliver a faster and more powerful response following a second encounter with an invading pathogen, effectively provides the immune system with ‘memory’. The exploitation of the immune system's memory is the basis for all long-term immunoprophylactic drugs (e.g. vaccines) and remains the goal of much long-term immunotherapeutic drug development.
In order for cells to perform their functions within the complex systems of an animal, the cells need to have ‘receptors’ on their surfaces. These receptors are capable of ‘recognising’ specific substances that control various essential processes such as activation, proliferation and adherence to other cells or substrates. For example, in the case of the immune system, the receptors on T and B cells allow them not only to recognise antigen but also to interact with each other and thus regulate their activities. Without these receptors, the cells would lack an essential means of communication and would be unable to act effectively in the concerted way that is essential for the immune system of a multicellular organism.
In order to be able to specifically recognise and deal with the wide range of pathogens present in the environment, the immune system has developed two types of highly variable antigen receptor on lymphocytes: antibodies in B cells and T cell receptors, or TCRs, in T cells.
There are a great many different possible antigen receptors present in the body, to enable the immune system to recognise a wide variety of invading pathogens. In fact there are approximately 1012 different B cells and T cell receptors in an individual. Each individual B cell has only one type of receptor, and so to deal with a particular pathogen, a B cell having the ‘best fitting’ receptor for an antigen of that pathogen must be selected. This process is termed ‘clonal selection’. In theory, only a single clone may respond (a monoclonal response) or several (an oligoclonal response) or many (a polyclonal response) depending on the number of antigens/epitopes exhibited by the pathogen, and the specificity of the various selected B cells to these antigen/epitopes.
There is a major difference between the types of antigen that can be recognised by B cells and T cells. As far as it is known, only the receptors on the surface of B lymphocytes (i.e. antibodies) are capable of directly recognising antigens such as proteins on viruses and bacteria, or foreign molecules dissolved in body fluid. Antibodies can also be produced in a soluble form by the B cells when they are activated and develop into plasma cells. The antibodies are also termed immunoglobulins (abbreviated to Ig). T cell receptors, on the other hand, recognise only short peptides, also known as T cell epitopes, on the surface of cells of the body. These T-cell epitopes are produced by degradation of larger proteins that are either self (i.e. naturally occurring body proteins) or non-self (i.e. derived from foreign organisms infecting the body). Only those derived from foreign proteins, i.e. antigens, are normally capable of inducing an immune response in the body. Once produced, these epitopes are bound to a special type of molecule, the MHC (major histocompatibility complex) and the resulting complex is then presented on the cell surface for binding the T cell receptor.
It should be clear that due to the destructive nature of the immune response, the response has to act only against foreign pathogens, not against the body's own cells or proteins. Thus, the immune system needs to distinguish between ‘self’ and ‘non-self’. It has been proposed that although clones of lymphocytes reacting against self are produced, they are deleted before any reaction can occur. This process is termed ‘clonal deletion’. It has also been proposed that any self-reacting lymphocytes could be retained but only in a ‘switched-off’ state. This mechanism is termed ‘clonal anergy’. Whatever the process considered, it remains unclear what is the exact underlying mechanism allowing lymphoid tissues, such as the thymus, to identify individual T cell clones reacting against self from the pool of T lymphocytes reacting only against non-self. The present inventors have now investigated more fully the mechanism of self/non-self discrimination, which has led to the development of the present invention. The inventors have now established a method of predicting the immunogenicity of a substance such as a peptide, which has enabled quicker identification of immunogenic peptide sequences within large proteins.
It has been known for many years that the major histocompatibility complex (MHC) plays a key role in the immune system of animals. The MHC molecules enable T cells to recognise antigens, as has already been discussed above. There are three general types of MHC molecule, class I, class II and class III. Class I and class II MHC molecules are glycoproteins that are present on the surface of the cell, whilst class III are usually soluble molecules present inside the cell. There are a large number of different types of MHC molecule. For example in humans (where MHC is termed HLA, human leukocyte antigen) there are several hundreds of different alleles of the genes coding for MHC molecules, meaning that in the human population there are many different types of HLA. The MHC of different species is typically named according to different conventions, thus MHC for mouse is termed H-2, for rat RT1 and for rabbit RLA. The different gene regions coding for different MHC molecules in an individual are usually individually named, such as HLA-A, HLA-C etc. in humans.
The MHC molecule is a critical immune system molecule, since it is this molecule that presents the epitopes of the antigens to the immune system. For example, if a T cell is to respond to a particular pathogen, the pathogen must have a least one antigen (such as a protein) that has at least one epitope (such as a peptide portion of the protein) that can bind to an MHC molecule on the surface of a cell and thus interact with a T cell which binds to the MHC-peptide complex. Thus, the immune response is dependent on the ability of the MHC to bind to an epitope. If there is no epitope that the MHC will bind to, or if there is no T cell which will bind to the MHC-peptide complex, then no immune response will occur.
In respect of ‘self’ proteins, however, one of several epitopes may be able to bind to the MHC molecule and hence potentially induce an immune response. On these occasions a specific “signal” must be provided for the self-reacting lymphocyte clones to be deleted or “switched off”.
Since, as indicated above, both self and foreign (i.e. non-self) peptides can bind to MHC molecules, the binding of various peptides to MHC molecules has received particular scrutiny in the immunology field. Many investigations have sought to calculate or predict the strength of binding between certain MHC (particularly HLA and H-2) types and peptide sequences, to try to account for immune responses, or the lack of them (i.e. the “signal” required for discrimination between self and foreign). Examples of these include the following:    Altuvia Y, Schueler O, Margalit H. 1995. “Ranking potential binding peptides to MHC molecules by a computational threading approach”. J. Mol. Biol., 249:244-250.    Altuvia Y, Sette A, Sidney J, Southwood S, Margalit H. 1997. “A structure-based algorithm to predict potential binding peptides to MHC molecules with hydrophobic binding pockets”. Hum. Immunol. 58: 1-11.    G. E. Meister, C. G. P. Roberts, J. A. Berzofsky, A. S. De Groot, “Two novel T cell epitope prediction algorithms based on MHC-binding motifs; comparison of predicted and published epitopes from Mycobacterium tuberculosis and HIV protein sequences” Vaccine, 13:581-591, (1995).    Gulukota K, Sidney J, Sette A, DeLisi C. 1997. “Two complementary methods for predicting peptides binding major histocompatibility complex molecules”. J. Mol. Biol. 267:1258-1267.    Pamer E G, Harty J T, Bevan M J. “Precise prediction of a dominant class I MHC-restricted epitope of Listeria monocytogenes”. Nature 1991; 353: 852-855.    Parker K C, Bednarek M A, Coligan J E. 1994. “Scheme for ranking potential HLA-A2 binding peptides based on independent binding of individual peptide side-chains”. J. Immunol. 152:163-175.    Rammensee H G, Friede T, Stevanoviic S. 1995. “MHC ligands and peptide motifs: First listing”. Immunogenetics 41:178-228.    Ruppert J, Sidney J, Celis E, Kubo R T, Grey H M, Sette A. 1993. “Prominent role of secondary anchor residues in peptide binding to HLA-A2.1 molecules”. Cell 74:929-937.    Schueler-Furman O, Elber R, Margalit H. 1998. “Knowledge-based structure prediction of MHC class I bound peptides: A study of 23 complexes”. Fold Des. 3:549-564.    Sette A, Buus S, Appella E, Smith J A, Chesnut R, Miles C, Colon S M, Grey H M. 1989. “Prediction of major histocompatibility complex binding regions of protein antigens by sequence pattern analysis”. Proc. Natl. Acad. Sci. USA 86:3296-3300.    Sette A, Sidney J, del Guercio M F, Southwood S, Ruppert J, Dahlberg C, Grey H M, Kubo R T. 1994a. “Peptide binding to the most frequent HLA-A class I alleles measured by quantitative molecular binding assays”. Mol. Immunol. 31:813-822.    Sette A, Vitiello A, Reherman B, Fowler P, Nayersina R, Kast W M, Melief C J M, Oseroff C, Yuan L, Ruppert J, et al. 1994b. “The relationship between class I binding affinity and immunogenicity of potential cytotoxic T cell epitopes”. J. Immunol. 153:5586-5592.    Stefan Stevanovic (2002): “Structural basis of immunogenicity”, Transplant Immunology 10 133-136    Sturniolo T, Bono E, Ding J, Raddrizzani L, Tuereci O, Sahin U, Braxenthaler M, Gallazzi F, Protti M P, Sinigaglia F, Hammer J. 1999. “Generation of tissue-specific and promiscuous HLA ligand databases using DNA microarrays and virtual HLA class II matrices”. Nat. Biotechnol. 17:555-561.    T. Sudo, N. Kamikawaji, A. Kimura, Y. Date, C. J. Savoie, H. Nakashima, E. Furuichi, S. Kuhara, and T. Sasazuki, “Differences in MHC Class I self peptide repertoires among HLA-A2 subtypes.” J. Immunol.: 155: 4749-4756, (1995).    T. Tana, N. Kamikawaji, C. J. Savoie, T. Sudo, Y. Kinoshita, T. Sasazuki, “A HLA binding motif-aided peptide epitope library: A novel library design for the screening of HLA-DR4-restricted antigenic peptides recognized by CD4+ T cells.” J. Human Genet., 43:14-21 (1998).    K. Falk, et al. “Allele-specific motifs revealed by sequencing of self-peptides eluted from MHC molecules”, Nature, Vol. 351, 290-297 (1991).    T Elliott et al. “Peptide-induced conformational change of the class I heavy chain”, Nature, Vol. 351, 402-407, (1991).    P. Parham, “Deconstructing the MHC”, Nature, Vol. 360, 300-301, (1992).    Hwai-Chen Guo et al., “Different length peptides bind to HLA-Aw68 similarly at their ends but bulge out in the middle”, Nature, Vol. 360, 364-367, (1992).    Y. Chen et al. “Naturally processed peptides longer than nine amino acid residues bind to the class I MHC molecule HLA-A2.1 with high affinity and in different conformations”, J. Immunol., 152, 2874-2881, (1994).    D. F. Hunt et al. “Characterization of peptides bound to the class I MHC molecule HLA-A2.1 by mass spectrometry”, Science, Vol. 255, 1261-1263, (1992).
Generally, the prior art attempts to predict the immunogenicity of particular peptides by calculating the strength of binding between that peptide and the known binding environment of a particular MHC molecule. The binding environment involves a ‘pocket’ in the MHC molecule that is adapted to accept a peptide of a certain length (such as 7-15 amino acids). The structure of the pocket may already be known from previous X-ray crystallographic studies. This strength may be calculated mathematically using appropriate algorithms for atomic and molecular interaction. Alternatively, the prior art may attempt to ‘score’ the binding strength of a peptide based upon motifs existing in the peptide, such as particular amino acids being present at particular positions in a peptide of a certain length, e.g. a proline present at position 3 in an 8-amino acid peptide binding to a particular known HLA molecule. Generally these approaches have met with limited success.